Project Summary Kallmann Syndrome (KS) is characterized by infertility and anosmia due to deficiency in gonadotropin- releasing hormone (GnRH) neuronal migration and olfactory bulb dysgenesis. Genetic studies have revealed that KS is caused by loss-of-function mutations in several genes including the prokineticin receptor 2 gene (PROKR2) observed in 10% of KS patients. Mice with global deletion of Prokr2 replicate the phenotype of KS patients displaying olfactory bulb dysgenesis, impaired GnRH neuronal migration and infertility. Whereas the role of PROKR2 during development is defined, little is known about PROKR2 neurons in adult reproduction. PROKR2 mRNA and the mRNA for its ligand are highly expressed in reproductive control sites of the adult mouse brain. Despite normal GnRH neuronal migration, heterozygous mice for Prokr2 loss of function mutation display longer estrous cycles compared to wild type littermates. Similarly, humans with PROKR2 mutations have a normal olfactory system but decreased fertility suggesting reproductive deficits independent of normal olfactory bulb morphogenesis and GnRH neuronal migration. However, the neural basis for PROKR2 role in adult reproduction is unknown. We recently developed a PROKR2-Cre mouse model in which Cre recombinase is driven by the Prokr2 promoter. With this mouse model, we mapped the distribution of PROKR2 expressing cells in the brain of both sexes. PROKR2 mRNA and GFP+ cells were highly expressed in subdivision of the medial amygdala in a sexually-dimorphic pattern. Male mice have higher PROKR2-Cre in the amygdalohippocampal area (AHi, also called posterior nucleus of the amygdala) whereas females have higher PROKR2 in the posterodorsal subdivision of the medial nucleus of the amygdala (MeApd). Both the MeApd and the AHi show dense expression of sex steroid receptors and play important role in reproductive function and associated behaviors. We hypothesize PROKR2-Cre neurons of the MeApd are necessary for typical control of female reproductive function (including estrous cyclicity), whereas PROKR2-Cre neurons in the AHi have a role in male reproduction. In two aims, we propose to map the neuronal projections of MeApd PROKR2 neurons in females and AHi PROKR2 neurons in males. We will also determine if PROKR2 neurons respond to opposite sex odors. Lastly, we will use chemogenetic technology, i.e. the designer receptors exclusively activated by designer drugs (DREADDs) to activate or inhibit PROKR2 neurons of the MeApd in females and of the AHi in males. We will determine if activation of these neurons increases circulating reproductive hormones and if inhibition of these neurons blocks the rise in reproductive hormones observed after exposure to opposite sex odors. Our studies will define a role for PROKR2 neurons in subdivisions of the medial amygdala, an important site of socio-sexual inputs and reproductive neuroendocrine responses in rodents and primates, including humans. Upon completion, we expect our studies will contribute to the understanding of the reproductive deficits associated with PROKR2 mutations.